WO1999053024A1 - Procedes d'identification de composes pouvant modifier une fonction mitochondriale - Google Patents

Procedes d'identification de composes pouvant modifier une fonction mitochondriale Download PDF

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WO1999053024A1
WO1999053024A1 PCT/US1999/008117 US9908117W WO9953024A1 WO 1999053024 A1 WO1999053024 A1 WO 1999053024A1 US 9908117 W US9908117 W US 9908117W WO 9953024 A1 WO9953024 A1 WO 9953024A1
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mitochondrial
cell
cells
fluorescence
compounds
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PCT/US1999/008117
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English (en)
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Eduardo Marban
Brian O'rourke
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Johns Hopkins University
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Priority to AU35592/99A priority Critical patent/AU3559299A/en
Publication of WO1999053024A1 publication Critical patent/WO1999053024A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria

Definitions

  • the present invention relates to methods for screening for compounds that modulate mitochondrial function by affecting mitochondrial redox potential. Methods of the invention also can be used to test for mitochondrial fitness.
  • mitochondria which produce most of the energy used by the body.
  • Energy derived from the utilization of substrates is required in order to maintain the non-equilibrium state necessary to carry out the basic functions of the cell (e.g. contraction, secretion, electrical propagation, ion pumping, cell division).
  • the Krebs cycle a.k.a. the citric acid or tricarboxylic acid cycle
  • oxidative phosphorylation convert acetyl CoA into ATP, the energy storage molecule directly utilized by the energy consuming reactions in the cell.
  • Mitochondrial substrate oxidation is a multi-step process that includes a series of reactions which transfer electrons from the initial substrate to nicotinamide adenine dinucleotide (NAD+) to produce NADH which is then reoxidized by passing electrons through the electron transport chain to the ultimate electron acceptor oxygen.
  • NAD+ nicotinamide adenine dinucleotide
  • protons (H+) are pumped out of the mitochondrial matrix (an - 2 - intracellular aqueous compartment bounded by the inner and outer mitochondrial membranes between it and the cell's cytoplasm) across the mitochondrial inner membrane (IMM) resulting in the establishment of a proton gradient.
  • This proton concentration gradient together with the large membrane voltage generated by the active charge movement across the IMM, provide the driving force for proton movement back into the matrix (the protonmotive force) which will be utilized by a specific IMM protein (the mitochondrial ATP synthase) to convert ADP (adenosine diphosphate) to ATP (adenosine triphosphate). ATP thus produced is transported out of the mitochondria and is available to perform the work required by the cell.
  • the relative impermeability of the IMM to ion leak and the presence of energy conserving pumps and exchangers in the membrane allows for the efficient utilization of the protonmotive force for cellular energy production rather than expending it for reestablishing the IMM gradient.
  • the IMM contains a number of energy dissipating high conductance pathways for ion and/or solute movement.
  • the physiological role of some of these pathways is apparent, for example, the pyruvate transporter is required to import substrate into the matrix for oxidative phosphorylation; however, in other cases, the physiological importance of IMM ion conducting pathways is still unknown.
  • a well known example is the mitochondrial megachannel (MMC) or permeability transition pore (PTP).
  • this large non-selective channel When activated, this large non-selective channel rapidly de-energizes the mitochondrion and has been implicated in several pathophysiological states.
  • Other known high conductance pathways include the calcium uniport, the mitochondrial inner membrane anion channel, the mitochondrial uncoupling protein of brown fat mitochondria, and the mitochondrial ATP-sensitive potassium channel.
  • a number of electrogenic e.g., the adenine nucleotide transporter, the glutamate-aspartate transporter, the Na-Ca exchanger
  • proton- compensated electroneutral e.g., the glutamate, pyruvate, and malate-citrate transporters
  • electroneutral e.g., the malate-phosphate, malate-ketoglutarate, carnitine, ornithine, and neutral amino acid transporters
  • the mitochondria are essential for efficiently providing ATP for carrying out the myriad functions of the cell, particularly in tissues with a high energy demand, such as muscle and brain. Consequently, defects in mitochondrial energy metabolism are usually associated with significant functional deficits or death.
  • the pathophysiologies related to mitochondrial dysfunction can be either primary or secondary.
  • primary mitochondrial diseases a genetic defect (either inborn or acquired) in a mitochondrial protein may lead to the incorrect assembly or catalytic activity of the protein, thus disrupting or impairing the entire biochemical pathway.
  • Secondary mitochondrial disorders may arise from the accumulation of toxic products within the cell (including oxygen free radicals), the accumulation of inhibitory metabolites, or lack of cofactors required for mitochondrial metabolism.
  • Mitochondrial cytopathies of differing origin often lead to similar clinical symptoms.
  • the disorders are first expressed in the most metabolically active tissues. They may present as muscle weakness and fatigue, mild muscle ache, or severe (and sometimes lethal) lactic acidosis during exercise.
  • these muscle deficiencies are also associated with central nervous system disorders, referred to as mitochondrial encephalomyopathies (e.g, KSS, Kearnes-Sayre syndrome; MERRF, myoclonus epilepsy with ragged red fibres; MELAS, mitochondrial encephalomyopathy/ lactic acidosis/stroke).
  • KSS Kearnes-Sayre syndrome
  • MERRF myoclonus epilepsy with ragged red fibres
  • MELAS mitochondrial encephalomyopathy/ lactic acidosis/stroke
  • These disorders may arise from point mutations in or deletions of large segments of mitochondrial DNA.
  • the specific enzyme affected is known (e.g.
  • Mitochondrial cytopathies can be classified by the site of the defect in mitochondrial oxidation. Defects in substrate transport (e.g. carnitine or carnitine- palmitoyl-transferase deficiencies), substrate metabolism (e.g. deficiencies in pyruvate dehydrogenase, pyruvate carboxylase, fatty acid oxidation, or organic acid metabolism), Krebs cycle activity (e.g. defects in oxoglutarate dehydrogenase or fumarase), the respiratory chain (NADH-Q reductase or cytochrome deficiencies), or energy coupling (ATP synthase defect, mitochondrial uncoupling diseases).
  • substrate transport e.g. carnitine or carnitine- palmitoyl-transferase deficiencies
  • substrate metabolism e.g. deficiencies in pyruvate dehydrogenase, pyruvate carboxylase, fatty acid oxidation, or organic acid metabolism
  • Krebs cycle activity e.g. defects in
  • mitochondrial metabolism has been suggested as an underlying cause of diseases associated with aging, including Alzheimer's and diabetes mellitus. Furthermore, mitochondria are the site of initiation of programmed cell death (apoptosis) and probably are the key factor in determining whether or not a cell will recover from an ischemic insult or proceed to necrosis.
  • mitochondria are central to the survival and function of the cell under normal conditions and play a major role in adapting to environmental stress.
  • the present invention relates to methods of assaying for mitochondrial function and more particularly to methods of identifying compounds that selectively modulate mitochondrial function.
  • the present invention relates to methods of detecting compounds that can positively impact mitochondrial function and increase cell energy output.
  • the invention relates to methods of detecting compounds that can decrease mitochondrial function in diseased cells.
  • the present - 5 - invention has a variety of useful applications including use in screens to detect compounds that can enhance overall health and fitness.
  • the invention includes methods for detecting the effects of agents acting on mitochondrial metabolism in intact cells by utilizing endogenous redox potential sensitive fluorophores located in the mitochondria.
  • the methods of the invention can enable low cost high throughput screening of compounds which modify the functional state of mitochondria for therapeutic applications.
  • Methods of the invention are applicable to the discovery of agents which modify the activity of any of the steps in energy metabolism. For instance, an exemplary and preferred application detects mitochondrially active agents in intact cardiac cells.
  • the methods of the present invention are useful for detecting drugs that alter mitochondrial function.
  • the invention provides a drug detection assay by measuring endogenous fluorescence in intact cells. In a normal oxygenated medium, the mitochondrial matrix is significantly reduced. Drugs that decrease the membrane potential across the inner mitochondrial membrane cause oxidation of the matrix, which is detected as a change in endogenous fluorescence by the methods of the present invention. Accordingly, the methods of the present invention are well-suited to detect compounds that can selectively enhance or decrease mitochondrial function.
  • cells are cultured and illuminated at wavelengths suitable to excite endogenous fluorescence.
  • the fluorescence is due to changes in the redox state of endogenous molecules located in the mitochondria.
  • These endogenous molecules function as reporters of mitochondrial oxidation state.
  • Preferred molecules include endogenous proteins that comprise fluorescent molecules such as a flavin moiety, or endogenous fluorescent molecules such as NAD.
  • One aspect of the invention relates to a method for identifying a compound capable of modulating mitochondrial function comprising contacting a eukaryotic cell with one or more candidate compounds and detecting a change in the mitochondrial redox state.
  • endogenous fluorescence of the cell mitochondria is indicative of a change of redox state.
  • the change in the redox state is an increase or decrease in the state of the mitochondria oxidation. That change is typically related to a suitable control assay as described below.
  • the fluorescence is measured of a nicotinamide adenine dinucleotide (NAD) or a flavin adenine dinucleotide (FAD) moiety, such as a protein comprising a linked flavin adenine dinucleotide (FAD) moiety.
  • NAD nicotinamide adenine dinucleotide
  • FAD flavin adenine dinucleotide
  • the FAD- linked protein is linked to a protein component of a mitochondrial redox pathway.
  • detection of the mitochondrial redox state further comprises measuring a change in fluorescence of an NAD molecule or FAD-linked enzyme, and correlating that change to a control assay comprising a mitochondrial oxidizing or reducing agent.
  • Illustrative oxidizing agents include dinitrophenol, and illustrative reducing agents include cyanide.
  • the cell is contacted with a plurality of candidate compounds or a library of candidate compounds.
  • the steps of contacting a eukaryotic cell with one or more compounds and detecting the change in the mitochondrial redox state of the cell are performed a number of times substantially simultaneously. These steps can be performed e.g. in a multi-well plate.
  • the eukaryotic cell used in certain methods of the present invention comprises a cardiac cell or a precursor cell thereof.
  • the eukaryotic - 7 - cell is a cardiac cell or precursor cell thereof that is immortalized.
  • the cardiac cell or precursor cell thereof is a primary cell.
  • the cell may comprise a ventricular myocyte or a skeletal myoblast.
  • the present invention relates to methods for detecting many different types of drugs capable of modulating mitochondrial function.
  • the present invention also relates to methods wherein the candidate compound activates a mitochondrial K ATP channel. Further, it relates to methods of assaying the activity of mitoK A ⁇ p channels using fluorescence methods. In certain prefened embodiments, the compound does not substantially activate a sarcolemmal K ATP channel.
  • the cell is contacted with the candidate compound(s) in vitro. In other embodiments, the cell is contacted with the candidate compound(s) in vivo. In yet other embodiments the cell is a tissue and the tissue is treated with the candidate compound ex vivo.
  • the present invention further relates to a method for detecting a compound capable of modulating mitochondrial redox potential, the method comprising: a) providing a population of eukaryotic cells; b) contacting a first portion of the cells with one or more candidate compounds; c) contacting a second portion of the cells with a known mitochondrial oxidizing or reducing agent; and d) measuring a difference between mitochondrial fluorescence produced in steps b) and c).
  • the cells are ventricular cells and the method further comprises measuring mitochondrial K A ⁇ p ion channel currents in those cells.
  • the compound activates a mitochondrial K A ⁇ p ion channel in the cells.
  • the method further comprises measuring sarcolemmal K A ⁇ p ion channel cunents in the cells.
  • the drug does not substantially activate the sarcolemmal K A ⁇ p ion channel currents at comparable concentrations.
  • the mitochondrial fluorescence is activated by a light at a wavelength of from about 250 to about 650nm.
  • the step of detecting or measuring is accomplished by fluorescence microscopy.
  • Prefened cells comprise detectable mitochondrial fluorescence.
  • such cells will often include cells from highly energetic tissues such as muscle and particularly cardiac and skeletal muscle cells.
  • certain rapidly dividing cells can also be used such as cancer cells (primary or cultured cell line) and immature cells (e.g., hemapoeitic cells).
  • the eukaryotic cells are selected from the group consisting of H9C2 (rat ventricular myocyte-derived cell line), AT-1, HL- 1 (atrial tumor derived cell line) and C212 (murine skeletal muscle-derived cell line) cells.
  • the methods identify a candidate compound drug that modulates mitochondrial oxidation (e.g. mitochondrial flavoprotein oxidation) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, in a mitochondrial redox assay of the invention relative to a control (i.e. the same assay where the candidate compound has not been exposed to the test cells) .
  • the EC 50 of identified candidate compounds is preferably no more than about 10 ⁇ M in a standard whole-cell-patch-clamp assay. - 9 -
  • the invention further relates to a method of detecting the activity of a mitochondrial ion channel or mitochondrial transporter comprising contacting a eukaryotic cell with one or more candidate compounds and detecting a change in the mitochondrial redox state as indicative of the activity of the ion channel or transporter.
  • the invention also further relates to drug compounds obtained by the above- described method.
  • Figures 1 (A) and (B) are graphs showing the effect of diazoxide on flavoprotein fluorescence and I ⁇ ⁇ TP , respectively.
  • Figure 2(A) is a graph showing the effect of 5-HD on the oxidative effect of diazoxide on flavoprotein and Figure 2(B) is a graph showing effect of 5-HD.
  • Figure 3 is a graph showing pooled data for fluorescence.
  • Figure 4 shows pooled data for I KATP .
  • Figure 5 is a graph showing a dose response curve for diazoxide.
  • Figures 6(A) and (B) show the effect of pinacidil on flavoprotein fluorescence and IKATP, respectively.
  • Figures 7(A) and (B) are graphs showing summarized data for percentage of flavoprotein oxidation and I KATP , respectively. - 10 -
  • Figure 8 are confocal images showing (A) a cell at baseline and (B) a cell after TMRE loading of the same cell. (Control), after 3 minute exposure to diazoxide (Diazo), after exposure to dinitrophenol (DNP) and cyanide (CN).
  • Diazo diazoxide
  • DNP dinitrophenol
  • CN cyanide
  • Figure 9 is a graph of pooled data showing protection of rabbit ventricular myocytes from ischemia by diazoxide.
  • One method of the present invention for detecting a compound capable of modulating mitochondrial function comprises contacting a eukaryotic cell with one or more candidate compounds and detecting a change in the mitochondrial redox state.
  • the present methods of identifying compounds that modulate mitochondrial function are more easily performed than previously known methods. Furthermore, the present methods involve minimal cost or expense to perform, as compared to other methods.
  • candidate compound refers to any chemical compound that can be added to a eukaryotic cell, and may comprise a compound that exists naturally within the cell or is exogenous to the cell.
  • the compounds include native compounds or synthetic compounds, and derivatives thereof. These compounds may also be refened to herein as “compound to be tested” or “compounds of interest”.
  • the compounds to be identified modulate the function of mitochondria preferably by eliciting a change in the redox state of the mitochondria.
  • redox state means the degree or level of oxidation or reduction of the matrix of the mitochondria at - 11 - any given time. In a normal oxygenated environment, the mitochondrial matrix is substantially in a reduced state. The redox state of the mitochondria can change as the result of many factors. As described above, certain disease states can alter the redox state of the mitochondria. In addition, certain compounds, or drugs, may alter the redox state of the mitochondria. For example, drugs that decrease the membrane potential across the inner mitochondrial membrane cause oxidation of the matrix.
  • any potassium-selective ion channels in the inner mitochondrial membrane would tend to dissipate the membrane potential established by the proton pump, (see e.g., Garlid, K.D., Biochimica et Biophysica Acta, 1996, 1275: 123-126). Such dissipation accelerates electron transfer by the respiratory chain, and leads to net oxidation of the mitochondrial matrix.
  • mitochondrial redox state is an important indicator of mitochondrial function.
  • Compounds that can affect the degree of mitochondria oxidation are generally refened to herein as "mitochondria modulating compounds" or other similar term.
  • the detection of the redox state of mitochondria can be accomplished by methods known in the art. Certain components within the mitochondria fluoresce in response to changes in the redox state of the mitochondria. Endogenous fluorescent molecules in the mitochondria comprise nicotinamide adenine dinucleotide (NAD/NADH), or molecules that comprise a flavin adenine dinucleotide (FAD) moiety.
  • NAD/NADH nicotinamide adenine dinucleotide
  • FAD flavin adenine dinucleotide
  • the degree of fluorescence, or change in fluorescence provides a measurement of the redox state of the organelle.
  • test cells The eukaryotic cells used in the methods of the present invention are sometimes refened to herein as “test cells” or “cells to be tested.”
  • the steps in the methods of the present invention can be performed in any one of a number of ways.
  • a population of cells to be tested can be divided into portions and the portions placed in the wells of multi-well plates, test tubes or eppendorf tubes, or other such holders as known in the art.
  • the contacting of the cells with one or more candidate compounds can be performed in numerous ways.
  • At least one portion of the cells to be tested are exposed to at least one candidate compound, while another portion of the cells acts as a control, without exposure to any compounds. If desired, another portion of the cells is exposed to a compound that is known to either reduce or oxidize the mitochondria. The numbers of portions of cells to be tested will depend on the number of candidate compounds. The mitochondria redox state of the cells is then measured.
  • the cells are exposed to an appropriate wavelength of light, selected to excite the fluorescent molecule.
  • an appropriate wavelength of light selected to excite the fluorescent molecule.
  • the amount of fluorescence of the cells exposed to the candidate compound is compared to the fluorescence of the control cells.
  • the difference in fluorescence is conelated to the change in the redox state of the mitochondria produced by exposure of the cells to the candidate compound as compared to the control cells.
  • the fluorescence of the test cells is measured prior to addition of one or more of the candidate compounds.
  • fluorescence is - 13 - then measured again after the cells are contacted with the candidate compounds.
  • fluorescence is measured during the course of contacting the cells, so that the change in fluorescence can be measured simultaneously with the contact.
  • the change in fluorescence is conelated to a change in the redox state of the mitochondria in response to the candidate compounds.
  • the cells may be exposed to one or more candidate compounds sequentially. That is, if the cells are divided into portions, each portion of test cells may be contacted with one compound and the fluorescence in response to that compound measured. The cells may then be washed by methods known in the art and then contacted with another candidate compound and fluorescence in response to that compound measured. These washing, contact and measuring steps can be repeated numerous times depending on the number of compounds to be tested. Alternatively, the washing step can be eliminated and the compounds added sequentially.
  • the cells can be contacted with a library of candidate compounds.
  • the conditions are optimized to provide a measurable and reversible change in fluorescence in response to known compounds, e.g., diazoxide and dinitrophenol (DNP).
  • DNP diazoxide and dinitrophenol
  • each compound in the library is placed in a well in a multi-well plate containing a portion of cells to be tested.
  • the compounds that produce the desired response e.g., increase in oxidation, can be selected based upon the degree of fluorescence, e.g., increase in fluorescence produced by the cells.
  • High-throughput screening involves the testing of a range of different chemical entities in biochemical assays. Any of the methods of identifying mitochondrial modulating compounds described herein can be used for high-throughput screening assays.
  • the "hits”, i.e., compounds that produce the desired response, found by any of the methods, can then be further characterized according to methods known in the art, e.g., using cellular physiology and imaging methods. - 14 -
  • the cells are further exposed to conditions that create or simulate a disease state.
  • a disease state For example, to test a compound for cardioprotective properties the cells are exposed to simulated ischemia and cell injury subsequent to treatment with one or more candidate compounds.
  • Such models are known in the art. An exemplary model is further described below and in the examples that follow.
  • the degree of prevention of the disease state is determined by comparing the fluorescence of the cells treated with candidate compounds with the fluorescence of untreated, diseased cells. Candidate compounds are selected if they prevent the degree of fluorescence exhibited by the control cells, i.e., the change in redox state caused by the disease.
  • test cells either have a disease state or normal test cells are subjected to conditions that create a disease state.
  • the diseased cells will have a certain degree of fluorescence based on the redox state created by the disease.
  • the cells are then contacted with candidate compounds and the change in fluorescence in response to the compounds is measured.
  • the compounds that reverse the fluorescence, i.e., the redox state of the cells, due to the disease can be selected.
  • the fluorescent molecule may comprise a protein which has a flavin adenine dinucleotide (FAD), or the fluorescent molecule may comprise nicotinamide adenine dinucleotide (NAD).
  • FAD flavin adenine dinucleotide
  • NAD nicotinamide adenine dinucleotide
  • the FAD-linked protein is an enzyme component of a mitochondrial redox pathway.
  • the wavelength used to excite the fluorescent molecule is preferably within from about 250 nm to about 650 nm, and most preferably at about 488 nm when the fluorescent molecule contains a FAD moiety.
  • the wavelength is suitably within from about 250 to about 450 nm, and more preferably from about 320 to about 400 nm, with about 360 nm being particularly prefened.
  • Fluorescence can be recorded using presently known methods and technology, e.g., standard or specialty fluorescent plate readers.
  • the fluorescent images can be analyzed by computer using software designed for such imaging, e.g., ImageTool (Univ. of Texas Health Sciences Center, San Antonio).
  • Fluorescence of NAD moiety can be detected according to methods known in the art, particularly those described in O'Rourke, B., et al., Science, 1994, Vol. 265, pp. 962-6. Fluorescence can also be detected by methods, such as, but not limited to fluorescent microscopy, photometry, and photographic film.
  • the method may comprise measuring fluorescence of an
  • NAD molecule or FAD-linked enzyme as a result of adding a candidate compound of interest and comparing that fluorescence to the fluorescence in a control assay that comprises a known mitochondrial oxidizing or reducing agent.
  • Useful oxidizing or reducing agents are known in the art.
  • Prefened oxidizing agents comprise dinitrophenol (DNP) and diazoxide.
  • DNP is a protonophore which uncouples respiration from ATP synthesis and collapses the mitochondrial potential and induces oxidation in the mitochondria.
  • a prefened reducing agent comprises cyanide. Cyanide inhibits cytochrome oxidase and thus stops electron transfer.
  • Other oxidizing and reducing agents can be readily selected for use in the control in accordance with the present disclosure.
  • the known mitochondrial oxidizing or reducing agent can be added to the test cells subsequent to addition of the candidate compounds.
  • the candidate compounds may or may not be washed from the test cells, depending on the assay protocol.
  • the compounds need not be washed from the cells for this assay to work.
  • one population of cells can be used to test a number of candidate compounds.
  • the cell contains numerous mitochondria, and a sufficient number to enable analysis in - 16 - accordance with the methods of the invention.
  • the cell comprises a cardiac cell or a precursor cell thereof. These cells may be immortalized or a primary cell. Examples of prefened cells comprise a ventricular myocyte or a skeletal myoblast. For high-throughput screening assays, as described below, the cells should be easy to produce and have good survival rates in culture. Such cell lines can readily be selected by one of ordinary skill in the art. Examples of useful cell lines include H9c2 cells, AT-1 cells and C2C12 skeletal myoblasts. The cells preferably will be selected to elicit a measurable and reversible change in fluorescence.
  • the methods of the present invention enable the identification of many different types of compounds capable of modulating mitochondrial function.
  • compounds that impact energy output generally e.g., glycolysis, oxidative phosphorylation, Krebs cycle, etc.
  • mitochondrial function is affected by the activity of many different channels and transporters in the mitochondrial membrane, e.g., potassium pumps, transporters and channels, proton pumps and proton channels. Compounds that affect the activity of any of these channels and transporters can be detected by the methods of the present invention.
  • the methods described herein are useful for identifying compounds that can be used to treat certain diseases and alter mitochondrial states.
  • Compounds that increase mitochondrial respiration or other mitochondrial output can be identified by the present methods by testing compounds for increasing mitochondrial respiration. Such compounds can be used to increase the overall fitness of an animal or improve the condition of a diseased tissue.
  • compounds that decrease the energy output of the mitochondria and therefore decrease the energy available to cells can be identified by the methods of the present invention.
  • Compounds that decrease mitochondrial respiration or other mitochondrial output are useful to decrease the division and spread of cancer cells.
  • the present methods can be used to identify compounds that are prophylactic, e.g., to induce or mimic ischemic preconditioning of cardiac muscle. - 17 -
  • Prefened drugs used to induce or mimic ischemic preconditioning act by affecting only mitO K ⁇ TP channels.
  • opening of mitochondrial K A ⁇ p channels dissipates the inner mitochondrial membrane potential established by the proton pump. This dissipation accelerates electron transfer by the respiratory chain, and if uncompensated by increased production of electron donors (such as NADH), leads to net oxidation of the mitochondria.
  • the methods of the present invention include the measurement of mitochondrial redox state by recording the fluorescence of FAD-linked enzymes in the mitochondria, to selectively screen for compounds that affect that redox state.
  • Prefened compounds for use on myocardial cells detected by the methods of the present invention activate a mitochondrial K AT p channel and reversibly oxidize the mitochondrial matrix, without having an effect on sarcolemmal K A ⁇ p channels.
  • diazoxide is an example of such a compound that can be detected using the present methods.
  • Diazoxide is a commonly used antihypertensive drug which causes dose-dependent mitochondrial oxidation and is cardioprotective. It is believed that diazoxide is an agonist of mitoK A ⁇ p channels.
  • the methods of the present invention can also be used to detect compounds that block oxidation induced by other chemicals.
  • the candidate compounds are potential oxidation blockers and are added before, during or after addition of compounds that induce oxidation of the mitochondria.
  • 5-HD which has been shown to inhibit K A p - 18 - channels in sarcolemma (Notsu, T., et al., J. Pharmacol. Exp. Ther., 1992, 260: 702-708), and isolated mitochondria (Garlid, K.D., et al., Biophy.
  • 5-HD is widely used to block ischemic preconditioning and cardioprotection induced by K A ⁇ p channel openers. 5-HD is an effective blocker of mitochondrial K A ⁇ p channels.
  • prefened assays for testing compounds that activate mitochondrial K AT p channels utilize a cellular ischemia model.
  • cells are centrifuged into a pellet to simulate the restricted extracellular space and reduced oxygen supply during ischemia, sampled at designated time points and stained with a hypotonic (85 mOsm) trypan blue solution to test the osmotic fragility of the membrane.
  • hypotonic (85 mOsm) trypan blue solution See Vander Heide, R.S., et al, J. Mol. Cell Cardiol, 1990, 22: 165-181).
  • mitochondrial K A ⁇ p channels may serve as effectors of cardioprotection by K A ⁇ p channel openers and protect myocytes against ischemic damage. It is theorized that this occurs through the dissipation of mitochondrial membrane potential resulting in a decrease in the driving force for calcium influx through the calcium uniporter. It has been reported that inhibition of the mitochondrial calcium uniporter by ruthenium red protects hearts against ischemia and reperfusion injury, consistent with this hypothesis. (Miyamae, M., et al., Am. J. Physiol, - 19 -
  • the method further comprises measuring mitochondrial K AT p ion channel cunents in the cells used in the assay.
  • the candidate compounds used on cells of myocardial origin do not substantially activate a sarcolemmal K A ⁇ p channel.
  • Sarcolemmal K AT p ion channel cunents can be measured simultaneously with fluorescence in cells, e.g., ventricular cells, by methods known in the art, such as whole cell patch-clamp method, which is generally prefened. See the examples for suitable procedures for this method. References herein to a "standard whole-cell patch assay" are intended to refer to the protcol described in the examples below. Changes in channel cunents can be measured in response to the addition of drugs to the cells.
  • prefened drugs that are to be used to induce cardioprotection do not substantially change the sarcolemmal K A ⁇ p ion channel cunents.
  • compounds identified by the methods of the invention include those that exhibit at least about a 100- fold greater activation of mitochondrial K AT p channels relative to activation of sarcolemmal K AT p channels, more preferably about a 500-fold greater activation of mitochondrial K A ⁇ p channels relative to activation of sarcolemmal K A ⁇ p channels, still more preferably about a 1000-fold greater activation of mitochondrial K A ⁇ p channels relative to activation of sarcolemmal K A ⁇ p channels.
  • an example of a use of this assay includes the identification of compounds that induce or mimic ischemic preconditioning by increasing oxidation of the mitochondria.
  • the methods of the present invention identify that increase mitochondrial oxidation.
  • the methods of the present invention can be used to identify compounds that affect, e.g., the numerous transporters and channels in mitochondria. While the methods have been described above with particular attention to mitochondrial K A ⁇ p channels, the methods can be applied to any channel or transporter.
  • the candidate compounds are contacted with the test cells as described above. If the candidate compound changes the redox state of the mitochondria, such change will be detected, e.g., by a change in fluorescence, and a mitochondrial modulating compound will be identified.
  • the effect of these compounds on the redox state can be measured simultaneously with the effect of the compounds on the activity of other channels or transporters through the use of methods described herein, e.g., whole- cell patch clamp.
  • the compounds identified by the methods of the present invention are useful as additions to enhance cell vitality in cell culture and in vitro assays.
  • the test cells comprise cells in a tissue of interest.
  • the tissue of interest is treated with the one or more candidate compounds ex vivo.
  • the tissue of interest or cells from the tissue (sometimes known as primary cells), is removed from a host organism.
  • the cells are then used in the methods described above. That is, the cells are contacted with one or more candidate compounds and the change in the mitochondria redox state of those cells is detected. Subsequently, the treated cells, i.e., oxidized cells, can be implanted back into the recipient host organism. - 21 -
  • the candidate compounds are contacted to the test cells in vivo.
  • the in vivo assays of the invention are particularly useful for subsequent evaluation of mitochondrial modulating compounds exhibiting suitable activity in an in vitro assay.
  • an animal model of cardiac muscle damage accompanying ischemia or an invasive surgical procedure such as balloon angioplasty is useful.
  • One suitable protocol involves administering to the animal a suitable vehicle or vehicle combined with one or more mitochondrial modulating compounds of interest.
  • the amount of the mitochondrial modulating compound administered will vary depending on several parameters including the extent of damage associated with the ischemia or surgical procedure of interest.
  • the animal will typically receive a candidate compound in a dose (e.g., i.m.
  • a prefened dosage schedule provides for administration of the compound starting 24 hours prior to conducting an invasive surgical procedure or inducing ischemia.
  • Daily injections, e.g., i.m. or i.p., of the compound are generally prefened. Subsequently, the animals are euthanized and the organ, e.g., heart removed for examination.
  • the term "invasive surgical procedure” means a medical or veterinary technique associated with significant damage to an organ such as the heart, liver or the kidney, or a limb.
  • the invasive surgical procedure can be associated with techniques involving, e.g., cardiac surgery, abdominothoracic surgery, arterial surgery, deployment of an implementation (e.g., a vascular stent or catheter), or endaterectromy.
  • the invasive surgical procedure is performed on a mammal such as a primate, particularly a human, rodent or a rabbit, or a domesticated animal such as a pig, dog or a cat. Ischemia can be induced in the animal by methods known in the art.
  • the compound is administered to the animal either as a sole active agent or in combination with other active compounds (e.g., 5-HD), or other candidate compounds to be tested.
  • activity of the candidate - 22 - compound in a given in vivo assay is compared to a suitable control (e.g., a sham- operated animal) in which the assay is conducted the same as the test assay but without administering the compound to the test subject.
  • a suitable control e.g., a sham- operated animal
  • test subjects can be employed, particularly mammals such as rabbits, primates, various rodents and the like.
  • the assays can be conducted in a wide variety of cells, tissues and organs.
  • the assays can detect useful mitochondrial modulating compounds by measuring the redox state of the mitochondria in several cell, tissue and organ settings.
  • the present invention further provides in vitro kits for detecting compounds capable of modulating mitochondrial function.
  • kits of the invention preferably include test cells and medium for use in the assay in an immediately usable or readily reconstituted form and preferably any other reagents necessary to ensure the activity and/or growth of the cells.
  • the kit includes a known mitochondrial oxidizing or reducing agent.
  • the kit further contains a detection device to facilitate determination of whether the candidate compound is a modulating compound.
  • the kit optionally contains multi-well plates or test tubes for running the assay.
  • the kit includes a vial or vessel containing test cells and an ampule or vial containing growth medium to sustain the test cells.
  • a kit may also include photographic film or other detection device.
  • the test cells are mixed with the growth medium and the candidate compounds are added. After a predetermined period of time, an aliquot of the assay mixture is spotted on or placed near the film, and the film is developed. The degree of spotting on the film indicates the redox state of the cells.
  • a control containing the test cells and growth medium, but not the candidate compound, is run simultaneously.
  • the film containing the cells contacted with the candidate sample will have a larger and/or darker spot than the control. If the candidate compound decreases the oxidative state, the control film will have a larger and/or darker spot than the cells treated with the candidate compound.
  • the difference in the spotting on the film for the mixture containing the candidate compound and the control is indicative of a change in the redox state of the cells in response to the candidate compound.
  • Collagenase type II was purchased from Worthington. Diazoxide was obtained from Sigma Chemical Co. Pinacidil and 5-hydroxydecanoic acid sodium (5HD) were purchased from Research Biochemical Int. Tetramethylrhodamine ethyl ester (TMRE) was obtained from Molecular Probes.
  • Diazoxide, pinacidil and TMRE were dissolved in DMSO before added into experimental solutions.
  • the final concentration of DMSO was less than 0.1%.
  • ventricular myocytes were isolated from adult rabbit hearts by conventional enzymatic dissociation, according to Liu, Y., et al., "Synergistic modulation of ATP-sensitive K + cunents by protein kinase C and adenosine: implications for ischemic preconditioning", Circ. Res., 1996, 78: 443-454, then washed several times with calcium-free solution. Calcium concentration was gradually brought back to 1 mM. Cells were then cultured on laminin-coated coverslips in Ml 99 culture medium with 5 % fetal bovine serum at 37°C. Experiments were performed over the next two days.
  • Endogenous flavoprotein fluorescence was excited using a xenon arc lamp with a bandpass filter centered at 480 nm, but only during the 100 msec step to -40 mV t to minimize photobleaching. Emitted fluorescence was recorded at 530 nm by a photomultiplier tube and digitized (Digidata 1200, Axon Instruments). (See Paucek, P., et al., "Cardioprotective effect of diazoxide and its interaction with mitochondrial K AT p: possible mechanism of cardioprotection", J. Mol Cell Cardiol, 1997, 29: A199(Abstract)). Relative fluorescence was averaged during the excitation window and calibrated using the values after dinitrophenol (DNP) and sodium cyanide (CN) exposure.
  • DNP dinitrophenol
  • CN sodium cyanide
  • Confocal images were obtained using a Diaphot 300 inverted fluorescence microscope with a PCM-2000 confocal scanning attachment (Nikon, Inc.). Fluorescence was excited by the 488 nm line of an argon laser and the emission at 505-535 nm was recorded. A time series of images was collected at intervals of -10 sec and baseline, diazoxide, DNP and CN images were enhanced by averaging 8-10 sequential images having stable mean fluorescence intensities during the exposure to each agent.
  • TMRE fluorescence was excited with the 535 nm line of a helium neon laser and recorded at greater than 605 nm.
  • a pseudocolor palette was applied to visualize the relative increase in mitochondrial flavoprotein oxidation state.
  • each cell suspension (0.5 ml) was placed in a 0.5 ml micro centrifuge tube and centrifuged for 20 seconds into a pellet. Each pellet occupied a volume of about 0.2 ml. Approximately 0.25 ml of excess supernatant was removed to leave a thin fluid layer above the pellet, and 0.2 ml of mineral oil was layered on the top of the pellet to exclude gaseous diffusion.
  • the small percentage of cells ( ⁇ 18 %) that were non- viable at the beginning of the experiment were mostly rounded and had been damaged as a known consequence of the enzymatic isolation process.
  • Mitra, R., et al. "A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates", Am. J. Physiol, 1985, 249: H1056-H1060.
  • Fig. 1 shows results from simultaneous measurements of flavoprotein fluorescence and membrane I KATP in cells exposed to diazoxide. The periods of drug treatment are marked with horizontal bars. Diazoxide (100 ⁇ M) induced reversible oxidation of the flavoproteins (Fig. 1A) but did not activate IKATP (Fig- 1 B).
  • the redox signal was calibrated by exposing the cells to DNP followed by CN at the end of the experiments.
  • DNP induced maximal oxidation
  • CN caused complete reduction of the flavoproteins ( Figure 1 A and Figure 2A).
  • membrane cunents were unchanged by diazoxide, I KATP eventually turned on after prolonged exposure to DNP ( Figures 1 B and 2B), indicating that these channels are operable under these experimental conditions despite the inability of diazoxide to open them.
  • Diazoxide (100 ⁇ M, DIAZO(l)) reversibly increased mitochondrial oxidation to 48 ⁇ 3 % of the DNP value (Figure 3).
  • Figure 3 shows DIAZO(l), first exposure to diazoxide; DIAZ0+5-HD(100), diazoxide in the presence of 100 ⁇ M 5-HD; DIAZO+5-HD(500), diazoxide in the presence of 500 ⁇ M 5-MD and DIAZ0(2), second exposure to diazoxide.
  • DNP exposure to dinitrophenol. The bar indicates the periods when cells were exposed to drug.
  • DIAZO(l) is the first exposure to diazoxide
  • DIAZ0+5-HD(100) means diazoxide in the presence of 100 ⁇ M 5-HD
  • DIAZO+5-HD(500) means diazoxide in the presence of 500 ⁇ M 5-MD
  • DIAZ0(2) means second exposure to diazoxide.
  • DNP indicates exposure to dinitrophenol. The bar indicates the periods when cells were exposed to drug.
  • the EC 50 for diazoxide to induce mitochondrial oxidation is 27 ⁇ M, as shown in Figure 5.
  • K A ⁇ p opener pinacidil
  • pinacidil Another K A ⁇ p opener, pinacidil, which opens sarcolemmal K A ⁇ p channels and is known to induce pharmacological preconditioning. Critz, S., et al., "Pinacidil but not nicorandil opens ATP sensitive K + channels and protects against simulated ischemia in rabbit myocytes", J. Mol Cell Cardiol, 1997, 29: 1123-1130.
  • pinacidil As shown in Figure 6A, pinacidil (100 ⁇ M) induced 35 ⁇ 8 % mitochondrial oxidation, comparable to the effect of diazoxide exposure in the same cell (41 ⁇ 5 %). Unlike diazoxide, pinacidil activated sarcolemnal I KATP (0.74 ⁇ 0.54 nA measured at O mV) in addition to inducing flavoprotein oxidation, (Figure 6 B) suggesting that pinacidil activates both mitochondrial and sarcolemmal K AT p channels. - 29 -
  • TMRE old Fig. 3B
  • the pattern of TMRE fluorescence was virtually identical to that of the flavoprotein fluorescence induced by diazoxide.
  • Figure 9 shows the fraction of cells killed by 60 or 120 min of ischemia as a percentage of the total number of viable cells prior to ischemia.
  • Cell killed (%) was calculated as number of cells killed by ischemia as a percentage of the total viable cells prior to ischemia.
  • the following designations have the following meanings: Cont, control.
  • Diazo 50 ⁇ M diazoxide.
  • 5-HD 100 ⁇ M 5-HD.

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L'invention concerne des procédés pour identifier un composé capable de moduler une fonction mitochondriale. Ces procédés consistent à placer une cellule eucaryote au contact d'un ou plusieurs composés candidats pour déceler une modification de l'état d'oxydoréduction de la cellule. L'invention concerne en outre des procédés dans lesquels une fluorescence endogène de la mitochondrie cellulaire indique une modification de l'état d'oxydoréduction.
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WO2001021758A2 (fr) * 1999-09-24 2001-03-29 The Johns Hopkins University Essai colorimetrique destine a des agents qui induisent un dysfonctionnement mitochondrial
WO2002040025A1 (fr) * 2000-11-17 2002-05-23 Orion Corporation Agents anti-inflammatoires
WO2002040026A1 (fr) * 2000-11-17 2002-05-23 Orion Corporation Nouvelle utilisation d'un derive de pyridazinone
EP3457132A1 (fr) * 2017-09-13 2019-03-20 IMG Pharma Biotech, S.L. Procédé de criblage de composés modulant l'activité de la chaîne de transport d'électrons

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US6183948B1 (en) * 1998-04-15 2001-02-06 Johns Hopkins University Methods to identify compounds affecting mitochondria
US6562563B1 (en) * 1999-11-03 2003-05-13 Mitokor Compositions and mehtods for determining interactions of mitochondrial components, and for identifying agents that alter such interactions
AU2001241420A1 (en) * 2000-01-14 2001-07-24 Mitokor Screening assays using intramitochondrial calcium
US6521617B2 (en) * 2000-10-13 2003-02-18 The Johns Hopkins University Treatment of apoptotic cell death
WO2004041256A2 (fr) * 2002-11-08 2004-05-21 Novo Nordisk A/S Decoupleurs chimiques surs pour le traitement de l'obesite
US7348135B2 (en) * 2003-08-28 2008-03-25 University Of Maryland Assay for detecting changes in mitochondrial membrane permeability and method of using same
EP2532390B1 (fr) 2005-02-16 2016-08-17 Md Bioalpha Co., Ltd. Composition pharmaceutique pour les maladies du foie.
EP1994168A4 (fr) * 2006-02-15 2009-05-27 Md Bioalpha Co Ltd Procédé de contrôle du ratio nad(p)/nad(p)h par une oxydoréductase
US9581600B2 (en) * 2010-12-09 2017-02-28 André Arsenault In vitro mitochondrial function test (MFT) and uses thereof

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US4808517A (en) * 1985-12-27 1989-02-28 Wisconsin Alumni Research Foundation Bioassay for toxic substances

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021758A2 (fr) * 1999-09-24 2001-03-29 The Johns Hopkins University Essai colorimetrique destine a des agents qui induisent un dysfonctionnement mitochondrial
WO2001021758A3 (fr) * 1999-09-24 2001-10-25 Univ Johns Hopkins Essai colorimetrique destine a des agents qui induisent un dysfonctionnement mitochondrial
US6479251B1 (en) 1999-09-24 2002-11-12 The Johns Hopkins University Colorimetric test for agents that induce mitochondrial dysfunction
WO2002040025A1 (fr) * 2000-11-17 2002-05-23 Orion Corporation Agents anti-inflammatoires
WO2002040026A1 (fr) * 2000-11-17 2002-05-23 Orion Corporation Nouvelle utilisation d'un derive de pyridazinone
US6878702B2 (en) 2000-11-17 2005-04-12 Orion Corporation Anti-inflammatory agents
EP3457132A1 (fr) * 2017-09-13 2019-03-20 IMG Pharma Biotech, S.L. Procédé de criblage de composés modulant l'activité de la chaîne de transport d'électrons
WO2019053137A1 (fr) * 2017-09-13 2019-03-21 Img Pharma Biotech, S.L. Procédé de criblage de composés modulant l'activité de la chaîne de transport d'électrons

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